Process of producing monoterpenes

ABSTRACT

The present invention relates to a process of producing a monoterpene and/or derivatives thereof. The process comprises the steps of: a) providing a host microorganism genetically engineered to express a bacterial monoterpene synthase (mTS); and b) contacting geranyl pyrophosphate (GPP) with said bacterial mTS to produce said monoterpene and/or derivatives thereof. The present invention also relates to a microorganism for use in producing a monoterpene and/or derivatives thereof and a recombinant microorganism adapted to conduct the step of converting geranyl pyrophosphate (GPP) into a monoterpene and/or derivatives thereof by expression of a bacterial mTS. It was shown to produce 1,8 cineole using 1,8 cineole synthase and to produce linalool using linalool synthase, both from  Streptomyces clavuligerus.

FIELD OF THE INVENTION

The present invention relates to a process of producing monoterpenes. Inparticular, the present invention relates to a process of producingmonoterpenes, for example in a host microorganism, by the action of abacterial monoterpene synthase.

BACKGROUND TO THE INVENTION

Terpenoids (also called isoprenoids) are the most abundant and largestclass (>75000) of natural products. Most commonly found in plants, theirbiological roles are multitude ranging from species to speciescommunication to intracellular signalling and defence against predatoryspecies. They have a wide range of applications and are used inpharmaceuticals, herbicides, flavourings, fragrances and biofuels. Dueto the broad commercial interest for terpenoids, efforts to synthesizethem by synthetic biology routes have gathered pace in recent years.

Terpenoid substrates are synthesized from the C5 isoprene buildingblocks, namely dimethylallyl pyrophosphate (DMAPP) and isopentenylpyrophosphate (IPP). Combination of DMAPP and IPP can generatesubstrates of varying carbon lengths, which can then be utilized byterpene synthases/cyclases to produce monoterpenes (C10), sesquiterpenes(C15), diterpenes (C20) and others. For example, geranyl pyrophosphate(GPP), the substrate for making all monoterpenes is synthesized bycoupling one molecule each of DMAPP and IPP.

Monoterpene synthases (mTS) are enzymes that use a single C10 substratemolecule geranyl pyrophosphate (GPP) to produce several thousand diversemonoterpenes. The structure of plant mTS has a two domain architecture:a class I terpenoid fold C-terminal domain and a relatively smallN-terminal domain whose function is unclear. The amino acid sequencevariations in the active site combined with conserved residues for GPPrecognition results in mTS carrying out some of the most complexreactions in biology leading to the formation of linear, monocyclic andbicyclic terpenoids.

Many monoterpene hydrocarbon scaffolds have been produced in engineeredmicrobes in recent years, using yeast or E. coli as a host and employingmTS from plant sources. However, the resulting monoterpene yields arerelatively low. Examples of monoterpenoids produced in such systemsinclude geraniol, β-myrcene, limonene and pinene.

E. coli is the workhorse for recombinant protein production around theworld in both academia and industry. This preferred choice is due to theease of introducing external DNA material into the cell, a fast growthcycle and the use of inexpensive growth media. As mentioned above, forthe production of monoterpenes using synthetic biology routes, plant mTShave been utilised. However, the use of such plant mTS has associateddisadvantages.

Experiments by the inventors have revealed that many plant mTS whenoverexpressed in E. coli, generate mostly insoluble protein i.e.,inactive material not suitable for monoterpene biosynthesis. Thislimited solubility has proved to be a bottleneck in the production ofmonoterpenes as the majority of the GPP molecules in the host cell arenot utilised for the synthesis of monoterpenes, resulting in low productyields/titres. This is particularly the case for biosynthesis oflinalool, which is widely used as perfume in cleaning agents. Plantlinalool synthases when employed in either yeast or E. coli result invery low product titres (0.1-1 mg/L_(org) ⁻¹). The presence of geranoidby-products (>10-fold excess) resulting from endogenous E. coli activityshows that substrate availability is not the cause of these observed lowtitres. Lack of robustness also makes plant mTS less attractive targetsfor protein engineering. In addition, mTS enzymes from plant sourcesoften show a high degree of product diversity resulting in productmixtures rather than clean product profiles. This is particularly thecase for the more complex bi-cyclic monoterpene scaffolds such as1,8-cineole (also called eucalyptol), which is used in flavourings,fragrances and cosmetics. Employing cineole synthases from severaldifferent plant sources all resulted in relative 1,8-cineole amounts of42-64% (Leferink, N. G. H. et al. A ‘Plug and Play’ Platform for theProduction of Diverse Monoterpene Hydrocarbon Scaffolds in Escherichiacoli. (2016)). The generation of single, clean products is desirable, asthis would require less downstream processing.

It is an object of the present invention to obviate or mitigate one ormore of the abovementioned problems.

SUMMARY OF THE INVENTION

The present invention relates to a process of producing monoterpenesand/or derivatives thereof. The present invention also relates tomicroorganisms for use in producing a monoterpene and/or derivativesthereof and recombinant microorganisms adapted to produce monoterpenes.

The invention is based in part on studies by the inventors in which theyshowed that the expression of certain bacterial monoterpene synthases(mTS) in E. coli results in a high yield of high purity monoterpenes.

The process of producing monoterpenes and/or derivatives thereofcomprises the steps of providing a host microorganism geneticallymodified to express a bacterial monoterpene synthase (mTS) andcontacting geranyl pyrophosphate with said bacterial mTS to produce saidmonoterpene and/or derivatives thereof.

In a first aspect of the invention there is provided a process ofproducing a monoterpene and/or derivatives thereof in a hostmicroorganism. The process comprises the following steps:

-   -   a) providing a host microorganism genetically modified to        express a bacterial monoterpene synthase (mTS); and    -   b) contacting geranyl pyrophosphate (GPP) with said bacterial        mTS to produce said monoterpene and/or derivatives thereof.

In studies undertaken by the present inventors, they have surprisinglyshown that it is possible to produce high yields of highly puremonoterpenes in host microorganisms when utilising bacterial monoterpenesynthases. As discussed in more detail below, the yield and purityobtained when using bacterial mTS was surprisingly much higher than whenplant-derived mTS were utilised.

Monoterpenes and/or Derivatives Thereof

The process of the present invention comprises contacting geranylpyrophosphate (GPP) with said bacterial mTS to produce said monoterpeneand/or derivatives thereof.

As will be appreciated by the skilled person, the term monoterpeneand/or derivative thereof is used to define a ‘product’ monoterpenedifferent to the ‘starting material’ geranyl pyrophosphate.

As described above, terpenes, for example monoterpenes have a variety ofapplications including in pharmaceuticals, herbicides, flavourings,fragrances and biofuels. The monoterpene produced in the process of thefirst aspect of the invention can be any suitable monoterpene orderivative thereof. Suitable derivatives of the monoterpenes of theinvention may be obtained by alkylation, oxidation or reduction forexample. For example, monoterpene derivatives may includemonoterpenoids.

As will be appreciated by the skilled person the monoterpene derivativesmay be produced by chemical derivitisation of the monoterpeneindependent of the mTS. Alternatively, the monoterpene derivatives maybe produced biologically by the mTS itself or another enzyme present inthe host microorganism.

The monoterpene or derivative thereof produced in the process of thefirst aspect of the invention may be geraniol, ocimene, citral,citronellal, citronellol, linalool, halomon, limonene, pinene, carene,sabinene, camphene, thujene, camphor, borneol, terpioline, terpinene,phellandrene, terpineol, fenchol, 1,8-cineole or β-myrcene for example.

In embodiments of the invention, the monoterpene may be linalool or1,8-cineole and/or derivatives thereof.

Host Microorganism

The host microorganisms of the present invention may be non-naturallyoccurring microorganisms for example genetically modified bacteria,archaea, yeast, fungus, algae or any of a variety of othermicroorganisms.

In embodiments of the invention, the host microorganisms are bacteria.Examples of suitable bacteria include enterobacteria belonging toproteobacteria of the genus Escherichia, Enterobacter, Pantoea,Klebsiella, Serratia, Erwinia, Salmonella or Morganella, coryneformbacteria belonging to the genus Brevibacterium, Corynebacterium orMicrobacterium and bacteria belonging to the genus Alicyclobacillus,Bacillus, Hydrogenobacter, Methanococcus, Acetobacter, Acinetobacter,Agrobacterium, Axorhizobium, Azotobacter, Anaplasma, Bacteroides,Bartonella, Bordetella, Borrelia, Brucella, Burkholderia,Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila,Clostridium, Coxiella, Ehrlichia, Enterococcus, Francisella,Fusobacterium, Gardnerella, Haemophilus, Helicobacter, Kelbsiella,Methanobacterium, Micrococcus, Moraxella, Mycobacterium, Mycoplasma,Neisseria, Pasteurella, Peptostreptococcus, Porphyromonas, Prevotella,Pseudomonas, Rhizobium, Rickettsia, Rochalimaea, Rothia, Shigella,Staphylococcus, Stenotrophomonas, Streptococcus, Treponema, Vibrio,Wolbachia or Yersinia.

Preferably the bacteria is of the genus Escherichia, preferablyEscherichia coli.

In embodiments in which the host microorganism is Escherichia coli (E.coli), the E. coli may be of one or more of K-12, B or W strains. Forexample, the host microorganism may comprise one or more of thefollowing strains: DH5α, DH10β, MG1655, W3110, DH1, MDS42, BL21 orMach1.

Examples of suitable yeasts or fungi include those belonging to thegenera Saccharomyces, Schizosaccharomyces, Candida, Kluyveromyces,Aspergillus, Pichia or Crytpococcus. In embodiments, yeast or fungispecies include those selected from Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromycesmarxianus, Aspergillus terreus, Aspergillus niger or Pichia pastoris,for example.

The host microorganism of the first aspect of the invention isgenetically modified (for example genetically engineered) to express abacterial monoterpene synthase (mTS).

As will be appreciated by the skilled person, the host microorganism inwhich the monoterpene is produced may be the same host microorganismwhich is genetically engineered to express a bacterial mTS.

The skilled person will appreciate that, in embodiments, the hostmicroorganism may be genetically modified to express a bacterialmonoterpene synthase not naturally encoded or expressed in the hostmicroorganism. The skilled person will further appreciate, that inembodiments, the host microorganism may be genetically modified toproduce more bacterial monoterpene synthase than a wildtypemicroorganism which encodes or expresses such a bacterial mTS.

Enhancing the production of bacterial mTS compared to a wildtypemicroorganism may include making modifications to existing nucleic acidsand/or proteins (for example by introducing a strong promoter before anormally silent or poorly expressed gene) by use of various geneticmodification techniques known in the art, as discussed further below.Enhancing the production of bacterial mTS compared to a wildtypemicroorganism may also include modifying the microorganism to expressone or more heterologous genes in the microorganism, for example a geneencoding a bacterial mTS from another microorganism, or genes which actto promote the functioning and expression of a bacterial mTS eitherdirectly or indirectly. For example, in embodiments, the hostmicroorganism may be E. coli which has been genetically modified toexpress a bacterial mTS from an alternative bacterial species, forexample Streptomyces.

The host microorganisms of the present invention may be modified toenhance production of monoterpenes and/or derivatives thereof. Forexample, the microorganisms may comprise modifications which decrease oreliminate the activity of an enzyme that catalyses synthesis of acompound other than monoterpenes by competing for the same substratesand/or intermediates (for example GPP). Alternatively, or in addition,the host microorganisms used in the process of the present invention maycomprise modifications that decrease or eliminate the activity of anenzyme which metabolises monoterpenes or intermediates in the productionof monoterpenes.

Enhancing the production of monoterpenes may include selecting hostmicroorganisms which are adapted to produce more monoterpene compared toa wildtype microorganism. In the context of the present invention, theterm ‘adapted’ when used in relation to a host microorganism means agenetically modified or engineered organism, or a mutant strain of anorganism, which has been selected on the basis that it expresses one ormore enzymes which result in enhanced production of monoterpenes.

In order to modify the activity of enzymes or proteins, mutations forincreasing, reducing or eliminating intracellular activities of theenzymes or proteins can be introduced into the genes of the enzymes orproteins by conventional random or site directed mutagenesis or geneticengineering techniques. Examples of the mutagenesis can include, forexample, X-ray or ultraviolet ray irradiation, treatment with a mutagen,in vitro site directed or random mutagenesis by the polymerase chainreaction. The site on the gene where the mutation is introduced can bein the coding region encoding the enzyme or protein or an expressioncontrol region such as a promoter. Examples of genetic engineeringtechniques include genetic recombination, transduction, cell fusion andgene knockouts.

Nucleic acid sequences can be introduced stably or transiently into ahost microorganism using techniques well known to the skilled person,including, for example, conjugation, electroporation, chemicaltransformation, transduction, transfection and ultrasoundtransformation.

Methods for constructing and testing the expression of a protein in amodified host microorganism can be performed using recombinanttechniques and detection methods well known to the skilled person, forexample as described in Sambrook and Russell, Molecular Cloning: ALaboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York(2001).

An expression vector or vectors can be constructed to include one ormore enzymes (for example a bacterial mTS) operably linked to expressioncontrol sequences functional in the microorganisms. Expression vectorsapplicable for use in the microorganisms of the invention include, forexample, plasmids, cosmids, phage vectors, viral vectors, episomes andartificial chromosomes, including vectors and selection sequences ormarkers operable for stable integration into a host chromosome.

Additionally, the expression vectors can include one or more selectablemarker genes and appropriate expression control sequences. Selectablemarker genes also can be included that, for example, provide resistanceto antibiotics or toxins, complement auxotrophic deficiencies, or supplycritical nutrients not in the culture media. Expression controlsequences can include constitutive, inducible or repressible promoters,transcription enhancers, transcription terminators or translationsignals for example.

Enhancement of the activity of an enzyme can include enhancingexpression of a target gene (for example a mTS) by replacing anexpression regulatory sequence of the target gene such as a promoter onthe genomic DNA or plasmid with a promoter which has an appropriatestrength. For example, the thr promoter, lac promoter, trp promoter, trcpromoter, pL promoter and tac promoter will be well known to the skilledperson. Examples of promoters with high expression activity inmicroorganisms such as bacteria can include promoters of the elongationfactor Tu (EF-Tu) gene, tuf, promoters of genes that encodeco-chaperonin GroES/EL and thioredoxin reductase, for example. Examplesof strong promoters and methods for evaluating the strength of promotersare well known in the art. Moreover, it is also possible to substituteseveral nucleotides in a promoter region of a gene, so that the promoterhas an appropriate strength.

In embodiments, the host microorganism may express (either naturally orby genetic modification) one or more enzymes which can convertisopentanyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) intogeranyl pyrophosphate (GPP). In embodiments, the one or more enzymes maybe a prenyltransferase under EC number 2.5.1.X, for examplegeranyl-diphosphate synthase under EC number 2.5.1.1. In embodiments,the prenyltransferase may be from Abies grandis, for example.

Therefore, in embodiments, the process may further comprise a step ofconverting IPP and DMAPP into GPP. Conversion may be achieved by theaction of a prenyltransferase as described above.

In embodiments, the host microorganism may express (either naturally orby genetic modification) one or more enzymes which can convert acetylCoA into IPP, for example by way of the mevalonate-dependent (MVA)pathway. In embodiments, the one or more enzymes may include, forexample, acetoacetyl-CoA thiolase (AtoB, EC 2.3.1.9) (from E. coli forexample), hydroxymethylglutaryl-CoA synthase (HMGS, EC 2.3.3.10) (fromS. cerevisiae for example), hydroxymethylglutaryl-CoA reductase (HMGR,EC 1.1.1.34) (from S. cerevisiae for example), mevalonate kinase (MK, EC2.7.1.36) (from S. cerevisiae for example), phosphomevalonate kinase(PMK, EC 2.7.4.2) (from S. cerevisiae for example), phosphomevalonatedecarboxylase (PMD, EC 4.1.1.33) (from S. cerevisiae for example) and/orisopentenyldiphosphate isomerase (idi, EC 5.3.3.2) (from E. coli forexample).

Therefore, in embodiments, the process may further comprise a step ofconverting acetyl CoA into IPP. Conversion may be achieved by the actionof one or more of the enzymes described above, for example.

Alternatively, or in addition, the host microorganism may express(either naturally or by genetic modification) one or more enzymes whichcan convert pyruvate into DMAPP, for example by way of themethylerythritol 4-phosphate (MEP) pathway. In embodiments, the one ormore enzymes may include, for example, 1-deoxyxylulose-5-phosphatesynthase (DXS, EC 2.2.1.7), 1-deoxyxylulose-5-phosphate reductoisomerase(DXR, IspC, EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphatecytidylyltransferase (YgbP, IspD, EC 2.7.7.60), 4-(cytidine5′-diphospho)-2-C-methyl-D-erythritol kinase (YchB, IspE, EC 2.7.1.148),(E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase (GcpE, IspG, EC1.17.7.1) and/or 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase(LytB, IspH, EC 1.17.7.4). In embodiments, such enzymes may be from E.coli, for example.

Therefore, in embodiments, the process may further comprise a step ofconverting pyruvate into DMAPP. Conversion may be achieved by the actionof one or more of the enzymes described above, for example.

In a further aspect of the invention there is provided a microorganismfor use in producing a monoterpene and/or derivatives thereof accordingto the process of the first aspect of the invention.

There is further provided a recombinant microorganism adapted to conductthe following step:

-   -   a) converting geranyl pyrophosphate (GPP) into a monoterpene        and/or derivative thereof by expression of a bacterial mTS.

The recombinant microorganism may be in accordance with the hostmicroorganism described in relation to the first aspect of theinvention.

In the context of the present invention, the term ‘recombinantmicroorganism’ is used to mean a genetically modified or engineeredorganism comprising genetic material which has been artificiallyconstructed and inserted into the organism. The genetic material maycomprise endogenous or heterologous nucleic acids.

The term ‘endogenous’ means deriving from the same species of organism.The term ‘heterologous’ means deriving from a different species oforganism.

There is yet further provided a microorganism genetically modified toexpress a bacterial mTS.

The microorganism may be in accordance with the host microorganismdescribed in relation to the first aspect of the invention.

Monoterpene Synthase

The process of the present invention comprises contacting geranylpyrophosphate (GPP) with a bacterial mTS to produce a monoterpene and/orderivatives thereof. The GPP is converted to the monoterpene and/orderivatives thereof by the action of the bacterial mTS.

As mentioned above, the present inventors have surprisingly demonstratedthat particularly high yields and purities of monoterpenes can beobtained when utilising bacterial mTS to convert GPP to monoterpenes.

As will be appreciated by the skilled person, the bacterial mTS of thepresent invention may be obtained from a bacterial species which is thesame as the host microorganism (i.e. the mTS may be endogenous). Forexample, the host microorganism may comprise E. coli and the bacterialmTS may be obtained from (i.e. originate from) E. coli.

Alternatively, the bacterial mTS may be obtained from a bacterialspecies different to the host microorganism (i.e. the mTS may beheterogenous). For example, the host microorganism may be a yeast or thehost microorganism may be E. coli and the bacterial mTS may be obtainedfrom a different bacterial species, for example Streptomyces.

The bacterial mTS may be a bacterial monoterpene synthase under ECnumber 4.2.3.x.

Sources of nucleic acids for genes encoding the bacterial mTS caninclude, for example, any species where the encoded gene product iscapable of catalysing the conversion of GPP to a monoterpene orderivative thereof. Exemplary bacterial sources include Escherichiacoli, Propionibacterium fredenreichii, Methylobacterium extorquens,Shigella flexneri, Salmonella enterica, Yersinia frederiksenii,Propionibacterium acnes, Bacillus cereus, Acinetobacter calcoaceticus,Acinetobacter baylyi, Acinetobacter sp., Clostridium kluyveri,Pseudomonas sp., Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonasfluorescens, Thermus thermophilus, Clostridium acetobutylicum,Clostridium cochlearium, Clostridium tetanomorphum, Clostridium tetani,Clostridium pasteurianum, Clostridium propionicum, Clostridiumsaccharoperbutylacetonicum Leuconostoc mesenteroides, Eubacteriumbarkeri, Bacteroides capillosus, Anaerotruncus colihominis,Natranaerobius thermophilus, Campylobacter jejuni, Corynebacteriumglutamicum, Bacillus subtilus, Serratia marcescens, Streptomyces speciesfor example Streptomyces coelicolor, Streptomyces cinnamonensis,Streptomyces avermitilis or Streptomyces clavuligerus, or Helicobacterpylori.

In preferred embodiments of the present invention, the bacterial mTS isderived from a Streptomyces species, for example Streptomycesclavuligerus. The present inventors have surprisingly shown that mTSderived from Streptomyces are particularly efficient at producingmonoterpenes with high purity and at high yield. In embodiments, thebacterial mTS is derived from Streptomyces clavuligerus.

In embodiments of the present invention, the bacterial mTS may comprise1,8-cineole synthase under EC number 4.2.3.108 and/or linalool synthaseunder EC number 4.2.3.25 or 4.2.3.26. As will be appreciated, in suchembodiments, the monoterpene produced comprises 1,8-cineole or linaloolrespectively.

In embodiments of the present invention, the bacterial mTS may comprise1,8-cineole synthase and/or linalool synthase derived from Streptomycesclavuligerus.

The bacterial mTS when utilised in the process of the first aspect ofthe present invention preferably produces a monoterpene yield (or titre)of at least 100 mg/L_(org) ⁻¹. More preferably, the bacterial mTSproduces a monoterpene yield of at least 150, 200, 250 or 300 mg/L_(org)⁻¹. Most preferably, the bacterial mTS produces a monoterpene yield ofat least 350 mg/L_(org) ⁻¹. The yield of monoterpene may be measured asdescribed in Leferink et al, 2016, for example by GC or GCMS andquantified during authentic standards. Yields are given as the amount ofproduct per litre of organic phase in which the products are captured.

As such, there is also provided the use of a bacterial mTS to improvethe yield (or titre) of a monoterpene and/or derivative thereof obtainedfrom GPP. In embodiments, the monoterpene is obtained from GPP in a hostmicroorganism. Suitably, the yield is improved compared to the yield ofa monoterpene and/or derivative thereof obtained from GPP using a plantmTS.

As will be appreciated, the bacterial mTS and host microorganism may bein accordance with those described in relation to the first aspect ofthe invention.

The bacterial mTS utilised in the process of the first aspect of theinvention preferably produces a monoterpene which is at least around 70%pure. When referring to purity, we refer to the amount of monoterpeneproduced from GPP, without the formation of by-products, for exampleother terpenes produced as mixed products (as described in Leferink etal, 2016).

Purities may be determined based on the monoterpenoids produced by themTS from GPP (as described in Leferink et al, 2016). For example, whenbacterial 1,8-cineole synthase is utilised as the mTS, small amounts ofα-terpineol and limonene may be produced in addition to the mainproduct, 1,8-cineole. In preferred embodiments, the bacterial mTSutilised produces a monoterpene which is at least around 75, 80, 85 or90% pure. More preferably the monoterpene produced is at least around95, 96, 97, 98 or 99% pure.

As such, there is also provided the use of a bacterial mTS to improvethe purity of a monoterpene and/or derivative thereof obtained from GPP.In embodiments, the monoterpene is obtained from GPP in a hostmicroorganism. Suitably, the purity is improved compared to the purityof a monoterpene and/or derivative thereof obtained from GPP using aplant mTS.

As will be appreciated, the bacterial mTS and host microorganism may bein accordance with those described in relation to the first aspect ofthe invention.

In embodiments of the invention, the bacterial mTS does not comprise aN-terminal α-barrel domain. Many plant mTS comprise N-terminal α-barreldomains. However, as described above, plant mTS when overexpressed in E.coli, generate mostly insoluble protein. Without wishing to be bound bytheory, the present inventors hypothesise that the additional domainpresent in many plant mTS may contribute to the insolubility of the mTSin the host microorganism. It may therefore be beneficial to utilise abacterial mTS which does not comprise a N-terminal α-barrel domain.

Further Process Steps

In the first aspect of the present invention, monoterpenes and/orderivatives thereof may be produced by the host microorganisms in afermentation medium. The host microorganisms of the present inventionmay be provided in a fermentation medium under conditions which saidhost microorganism will produce a monoterpene or derivative thereof.

In embodiments, the process of the first aspect of the inventioncomprises culturing the host microorganism in said fermentation medium.Culturing may require a carbon based feedstock from which themicroorganism may derive energy and grow.

The fermentation medium may be a surrounding medium which surrounds thehost microorganism. Preferably a carbon based feedstock is present inthe medium, for example dissolved or suspended in the medium or mixedwith the medium.

The fermentation medium may be any commercially available mediumsuitable for the needs of the host microorganism as will be well knownto the skilled person. The fermentation medium may comprise a carbonbased feedstock and a nitrogen source, as well as additional compoundsrequired for growth of the host microorganism, for example, antibiotics,buffers, phosphate, sulphate, magnesium salts etc.

Suitable carbon based feedstocks will be well known to the skilledperson working in this field of technology. The carbon based feedstockmay include, for example, glucose, maltose, sucrose, starch and/or maybe derived from wastes, for example food waste or wastes from industry,such as forestry or agriculture.

The amount of feedstock required will vary depending on the needs of thehost microorganism and the length of culturing of the hostmicroorganism, for example.

As will be appreciated by the skilled person, the host microorganism maybe genetically modified to produce GPP (as set out above) or the GPP maybe provided in the fermentation medium, for example.

The host microorganism may be cultured as a batch, fed-batch orcontinuous process. Preferably, the culturing is performed on anindustrial scale.

In embodiments, the process of the present invention may furthercomprise the step of removing the monoterpene and/or derivative thereoffrom the host microorganism, for example extracting the monoterpene fromthe host microorganism or fermentation medium in which the hostmicroorganism is cultured. For example, when the host microorganism ispresent in a fermentation medium, the process may comprise removing orextracting the monoterpene or derivative thereof from contact with thefermentation medium.

In embodiments, removing the monoterpene and/or derivative thereof maycomprise solvent extraction. For example, an organic phase may beprovided in contact with the fermentation medium. The organic phase mayinclude the monoterpene in a higher concentration than that in thefermentation medium. The organic phase may be an organic solvent, forexample. As will be appreciated by the skilled person, any suitableorganic solvent could be utilised, and may comprise nonane (for examplen-nonane), dodecane (for example n-dodecane), hexadecane (for examplen-hexadecane) or diisononyl phthalate.

Alternatively, the monoterpene may be removed from the fermentationmedium by steam distillation, under supercritical carbon dioxide or bypressurisation, for example.

The described and illustrated embodiments are to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiments have been shown and described and thatall changes and modifications that come within the scope of theinventions as defined in the claims are desired to be protected.Moreover, any one or more of the above described preferred embodimentscould be combined with one or more of the other preferred embodiments tosuit a particular application.

It should be understood that while the use of words such as“preferable”, “preferably”, “preferred” or “more preferred” in thedescription suggest that a feature so described may be desirable, it maynevertheless not be necessary and embodiments lacking such a feature maybe contemplated as within the scope of the invention as defined in theappended claims. In relation to the claims, it is intended that whenwords such as “a,” “an,” or “at least one,” are used to preface afeature there is no intention to limit the claim to only one suchfeature unless specifically stated to the contrary in the claim.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be further described with reference tothe following figures which show:

FIG. 1: GC-MS analysis using bacterial 1,8-cineole synthase (bCinS) andbacterial linalool synthase (bLinS). A) bCinS platform product profile.B) bCinS conversion of GPP (20 mM). C) 1,8-cineole standard (0.1 mg/ml).D) bLinS platform product profile. E) bLinS conversion of GPP (20 mM).F) R-linalool standard (0.1 mg/ml). G) cis- and trans-nerolidolstandards (0.1 mg/ml). IS=internal standard (sec-butyl benzene).

FIG. 2: Structure of bCinS-FNPP complex. Superposition of plant cineolesynthase (dark) and bCinS (light). The N-terminal and C-terminal domainsof the plant CinS are labelled. An arrow indicates the conformationaldifference in the helix-break motif.

Materials and Methods

Expression and Purification of Bacterial 1, 8-Cineole Synthase (bCinS)and Bacterial Linalool Synthase (bLinS)

The full-length genes coding for 1,8-cineole synthase (WP_003952918) andlinalool synthase (WP_0003957954) from Streptomyces clavuligerus ATCC27064 were codon optimized and synthesized from GeneArt (LifeTechnologies). The genes were amplified using PCR and sub-cloned intodouble digested (Ncol and Xhol) pETM11 vector using Infusion cloning(Clontech). The final construct coded for either 1,8-cineole synthase(bCinS) or linalool synthase (bLinS) with a 6X-His tag followed by a TEVprotease cleavage site at the N-terminus. The expression andpurification method explained below was identical for both the proteins.

The plasmid was transformed into ArcticExpress (DE3) cells (Agilent) andfew colonies were inoculated into 100 ml 2X-YT media containing 40 μg/mlof kanamycin and 20 μg/ml of gentamycin and grown for 3-4 hours at 37°C. The culture was diluted into 3 l of fresh 2×-YT media containing 40μg/ml of kanamycin and allowed to grow at 37° C. until the OD at 600 nmreached 0.6-0.8. At this stage, the temperature was reduced to 16° C.and 0.1 mM Isopropyl β-D-1-thiogalactopyranoside was added and incubatedfor 14-18 hours. The cells were harvested by centrifugation at 6000 gfor 10 minutes and the pellet was resuspended in buffer A (25 mM Tris pH8.0, 150 mM NaCl, 1 mM DTT, 4 mM MgCl₂ and 5% (v/v) glycerol). The cellswere lysed by sonication and the debris was removed by centrifugation at30,000 g for 30 minutes. The supernatant was filtered through a 0.2 μmfilter and loaded onto a 5 ml HisTrap column (GE Healthcare)pre-equilibrated with buffer A. The column was washed with buffer Acontaining 10 mM imidazole (pH 8.0) and increasing up to 40 mM imidazoleby step gradients with 3 column volume for each concentration.Increasing the concentration of imidazole to 200-500 mM eluted theprotein. The purified protein was desalted using Centripure P100 column(emp biotech) equilibrated with buffer A. To remove the His tag, TEVprotease was added (1:1000 (w/w)) to the protein and incubated at 4° C.with gentle mixing for 24 hours. The TEV protease was removed by passingthe protein mixture through a 5 ml HisTrap column and the flow throughwas collected. The His-tag removed protein was concentrated and loadedonto a Hiload Superdex (26/60) S75 column (GE Healthcare)pre-equilibrated with buffer A. Pure fractions from the gel filtrationcolumn were concentrated to 13-15 mg/ml and stored at −80° C. asaliquots.

Biotransformations

The 0.25 ml reactions were prepared using buffer A and setup in glassvials containing 2 mM GPP and 20 μM of bCinS or bLinS. The vials wereincubated at 25° C. with gentle shaking for 16 hours. The vials werecooled down to 4° C. and 0.25 ml of ethyl acetate containing 0.01%sec-butyl benzene as internal standard was added. The samples werevortexed for 2 min and then spun at 18,000 g for 5 min. The supernatantfractions containing the ethyl acetate layer were carefully removed anddried over anhydrous magnesium sulfate. The samples were analysed byGC-MS.

Linalool and 1,8-Cineole Production in E. coli

Both bLinS and bCinS genes, including RBS, were amplified from theirrespective pETM-11 expression vectors using primers pET_IF_Fw(5′-CATCCCCACTACTGAGAATC-3′) (SEQ ID No: 1) and pET_IF_Rv(5′-GGTGGTGGTGCTCGAGTTA-3′) (SEQ ID No: 2) and cloned using InFusion(Takara) into plasmid pGPPSmTS15, which was PCR linearised using theprimer pair Vector_IF_Fw (5′-TAACTCGAGCACCACCACCACC-3′) (SEQ ID No: 3)and Vector_IF_Rv (5′-TCAGTAGTGGGGATGTCGTAATCG-3′) (SEQ ID No: 4)resulting in plasmids pGPPSmTS38 and pGPPSmTSS39, respectively. Correctinsertion was confirmed by automated sequencing (Eurofins).

For monoterpenoid production the pGPPSmTS plasmids were co-transformedwith pMVA into E. coli DH5a and grown as described previously (Leferink,N. G. H. et al. A ‘Plug and Play’ Platform for the Production of DiverseMonoterpene Hydrocarbon Scaffolds in Escherichia coli. (2016)). Briefly,expression strains were inoculated in terrific broth (TB) supplementedwith 0.4% glucose in glass screw capped vials, and induced for 72 h at30° C. with 50 μM (isopropyl β-D-1-thiogalactopyranoside) IPTG and 25 nManhydro-tetracycline (aTet). A 20% n-nonane layer was added to capturethe volatile terpenoids products. After induction, the nonane overlaywas collected, dried over anhydrous MgSO₄ and mixed at a 1:1 ratio withethyl acetate containing 0.1% (v/v) sec-butyl benzene as internalstandard.

GC-MS Analysis

The samples were injected onto an Agilent Technologies 7890B GC equippedwith an Agilent Technologies 5977A MSD. The products were separated on aDB-WAX column (30 m×0.32 mm i.d., 0.25 μM film thickness, AgilentTechnologies). The injector temperature was set at 240° C. with a splitratio of 20:1 (1 μl injection). The carrier gas was helium with a flowrate of 1 ml min−1 and a pressure of 5.1 psi. The following oven programwas used: 50° C. (1 min hold), ramp to 68° C. at 5° C. min⁻¹ (2 minhold), and ramp to 230° C. at 25° C. min⁻¹ (2 min hold). The ion sourcetemperature of the mass spectrometer (MS) was set to 230° C. and spectrawere recorded from m/z 50 to m/z 250. Compound identification wascarried out using authentic standards and comparison to referencespectra in the NIST library of MS spectra and fragmentation patterns asdescribed previously (Leferink, N. G. H. et al. A ‘Plug and Play’Platform for the Production of Diverse Monoterpene Hydrocarbon Scaffoldsin Escherichia coli. (2016)).

Chemical Synthesis of FGPP and FNPP

A Horner-Wadsworth-Emmons reaction was performed by treating ethyl(diethoxyphosphoryl)fluoroacetate with sodium hydride followed by6-methyl-5-hepten-2-one resulting in a mixture of 2-fluoronerol and2-fluorogeranoil in almost equal ratio (1:1.08) with an 86% yield. Theisolated products were then treated with H₃PO₄(Et₃N)₂ salt to give thecorresponding mono and dephosphorylated products.

Crystallization of bCinS and bLinS

The crystallization trials containing 200 nl of protein and 200 nl ofprecipitant solution were setup in 3-well swissci plates using mosquitorobot (TTP Labtech). Five screens—Morpheus I and II, JCSG+, PACT premierand SG1 (Molecular Dimensions Ltd) were used for initial trails. ForbCinS and bLinS, three variants were screened—Apo, with 2 mM FGPP andwith 2 mM FNPP. The bCinS-FNPP crystallized in Morpheus II A4 condition(90 mM of LiNaK (0.3 M Lithium sulphate, 0.3 M Sodium sulphate, 0.3 MPotassium sulphate), 0.1 M of buffer system 4 (1 M MOPSO, 1 M Bis-Tris)pH 6.5, and 50% precipitant mix 8 (10% PEG 20000, 50% Trimethyl propane,2% NDSB 195)). The bLinS-FNPP crystallized in Morpheus D7 condition(0.12 M Alcohols (0.2 M 1,6-Hexanediol; 0.2 M 1-Butanol; 0.2 M1,2-Propanediol; 0.2 M 2-Propanol; 0.2 M 1,4-Butanediol; 0.2 M1,3-Propanediol) 0.1 M Buffer System 2 7.5 (1.0 M Sodium HEPES; MOPS(acid)) 50% v/v Precipitant Mix 3 (40% v/v Glycerol; 20% w/v PEG 4000)).LinS-apo crystallized in SG1 E2 condition (25% w/v PEG3350). AlthoughbCinS-apo crystallized, optimization of growth conditions failed toproduce single crystals of sufficient size for harvesting. ThebCinS-FNPP and bLinS-FGPP crystals were cryo-protected by soaking inmother liquor. The bLinS-apo crystals were cryo-protected by soaking inmother liquor supplemented with 20% glycerol. For FGPP and FNPPcomplexes, the ligands were included in the cryo-solution. The crystalswere harvested and cryo-cooled by plunging in liquid nitrogen.

Structure Solutions

The bLinS and bCinS X-ray datasets were collected at Diamond LightSource (DLS). The images were integrated and scaled by xia2 automateddata processing pipeline, using XDS and XSCALE. Crystals of bCinSbelonged to the triclinic system (spacegroup P1) and contained twomolecules in the asymmetrical unit (ASU), whereas bLinS crystalsbelonged to the tetragonal system (spacegroup 14) and also contained twomolecules in ASU. The bLinS structures (bLinS-apo and bLinS-FGPP) weresolved by molecular replacement using Pentalenene synthase structure(PDB 1PS1) as the search model in Phaser. The bCinS-FNPP structure wassolved by model replacement using the bLinS-apo structure as the searchmodel. The bLinS-apo, bLinS-FGPP and bCinS-F-NPP models were built usingAutobuild in Phenix. The structures were completed using iterativerounds of manual model building in coot and refinement in phenix.refine.The structures were validated using molprobity tools and PDB_REDO.

Plasmids

Table 1 below shows the plasmids used in this study.

TABLE 1 Plasmids used in this study Description (Origin of replication,Antibiotic marker, Reference(s), Plasmid Promoters reference Plasmidname and Operons) Source pMVA BbA5a-MTSAe- p15A, Kanr, LeferinkT1f-MBI(f)- PlacUV5, MTSA, et al, T1002i T1, MBI-f, T1002 2016pGPPSmTS15 pBbB2a- pBBR, Ampr, Ptet, Leferink trAgGPPS(co)-trAgGPPS(co)- et al, 2016 trSLimS_Ms trSLimS_Ms pGPPSmTS38 pBbB2a- pBBR,Ampr, Ptet, This study trAgGPPS(co)- trAgGPPS(co)- bLinS bLinSpGPPSmTS39 pBbB2a- pBBR, Ampr, Ptet, This study trAgGPPS(co)-trAgGPPS(co)- bCinS bCinS

Results

The present inventors undertook significant testing to determine howmonoterpenes might be produced in bacterial hosts with greaterefficiency. In particular, the experiments undertaken by the inventorssurprisingly identified a number of bacterial mTS, including bLinS andbCinS from Streptomyces clavuligerus, whose expression in an E. colisystem resulted in much higher monoterpene production, and in greaterpurity, than when plant mTS were utilised.

Linalool and 1,8-Cineole Production in E. coli

When expressed in E. coli, large quantities (>100 mg/litre) of bLinS andbCinS were produced and the enzymes were stable and soluble, whencompared to plant mTS which mostly resulted in insoluble or partiallysoluble material. Biotransformation reactions showed bLinS and bCinSproduced linalool and 1,8-cineole respectively when supplied with GPP.No by-products were observed when analyzed by GC-MS (FIG. 1).

To test for suitability in synthetic biology approaches, both bLinS andbCinS were inserted in an E. coli ‘plug-and-play’ monoterpenoidproduction platform, devised by the inventors, which consists of twogene modules (Leferink, N. G. H. et al. A ‘Plug and Play’ Platform forthe Production of Diverse Monoterpene Hydrocarbon Scaffolds inEscherichia coli. (2016)). The first module (pMVA) contains a hybrid MVApathway under regulation of IPTG-inducible promoters and the second(plasmid series pGPPSmTS, table 1) consists of a refactored,N-terminally truncated geranyl diphosphate synthase (GPPS) gene fromAbies grandis (AgtrGPPS2) followed by an mTS gene (for example bLinS orbCinS) under control of a tetracycline-inducible promoter.

Strains containing both pMVA and a pGPPSmTS plasmid were grown in atwo-phase shake flask system using glucose as the feedstock and n-nonaneas the organic phase. Products, which accumulated in the organic phase,were identified and quantified by GC-MS analysis.

Product profiles and titres obtained with bLinS and bCinS were comparedwith previously obtained product profiles using mTSs from plant sources(FIG. 1), i.e. LinS from Artemisia annua (RLinS_Aa) and CinS from Salviafruticosa (CinS_Sf), Arabidopsis thaliana (CinS_At), and Citrus unshiu(CinS_Cu) (Leferink, N. G. H. et al. A ‘Plug and Play’ Platform for theProduction of Diverse Monoterpene Hydrocarbon Scaffolds in Escherichiacoli. (2016)).

The inventors surprisingly found that both bacterial mTS outperformedthe plant enzymes. BLinS produced about 300-fold more linalool thanRLinS_Aa, 363.3±57.9 mg vs 1.3 mg L_(org) ⁻¹. Without wishing to bebound by theory, it is thought that this high yield can be attributed tothe high solubility of BLinS, compared to corresponding plant mTS.

Both bCinS and bLinS also produced much purer monoterpenes that plantmTS. bCinS produced 116.8±36.4 mg L_(org) ⁻¹ (96% pure) 1,8-cineolecompared to the plant enzymes: 118.2 mg L_(org) ⁻¹ (67% pure) forCinS_Sf; 46.6 mg L_(org) ⁻¹ (42% pure) for CinS_At; and 18.2 mg L_(org)⁻¹ (63% pure) for CinS_Cu.

In addition to the formation of GPP via the heterologous GPPS, E. colinatively produces the sesquiterpene precursor farnesyl diphosphate(FPP). Interestingly, plugged into the inventors platform, bLinS wasable to convert FPP to nerolidol (159.1±7.3 mg L_(org) ⁻¹), in additionto the formation of linalool from GPP, suggesting that bLinS acts asboth monoterpene and sesquiterpene synthase. No sesquiterpene productswere detected for bCinS under the specified conditions when plugged intothe platform.

Structure of bCinS Substrate Analog Complex and Comparison with PlantCineole Synthase

The inventors solved the bCinS structure in complex with a 2-fluoroderivative of GPP isomer (FNPP). The FNPP acts as a substrate inhibitorby blocking the ionisation step, which in turn stops the diphosphaterelease and formation of the geranyl cation. The structure revealed adimer with the active sites located in an anti-parallel fashion.

The present inventors then compared the structure of bCinS to thestructure of 1,8-cineole synthase from a plant in a ligand free state(PDB 2J5C). Compared to the plant enzyme, the bacterial enzyme lacks anN-terminal α-barrel domain (FIG. 2). Comparing the C-terminal domain ofthe plant enzyme (apo form) with bCinS-FNPP complex (sequence similarly25%) shows conformational changes around the active site. In the plantenzyme, the kink region of helix break motif of helix G, whichencompasses the residues described for induced-fit mechanism, isobserved to have the most movement where it is protruding inwardstowards the active site and almost reaching the location of thediphosphate in bCinS-FNPP complex (FIG. 2).

It will be appreciated that numerous modifications to the abovedescribed process, microorganisms and use thereof may be made withoutdeparting from the scope of the invention as defined in the appendedclaims. For example although the specific examples described havefocussed on the monoterpene synthases bCinS and bLinS from Streptomycesclavuligerus, it will be appreciated that other monoterpene synthasesfrom alternative bacterial species could be utilised.

Sequence listing SEQ ID No. 1: catccccact actgagaatc 20 SEQ ID No. 2:ggtggtggtg ctcgagtta 19 SEQ ID No. 3: taactcgagc accaccacca cc 22SEQ ID No. 4: tcagtagtgg ggatgtcgta atcg 24

1. A process of producing a monoterpene and/or derivatives thereof in ahost microorganism comprising the following steps: (a) providing a hostmicroorganism genetically modified to express a bacterial monoterpenesynthase (mTS); and (b) contacting geranyl pyrophosphate (GPP) with saidbacterial mTS to produce said monoterpene and/or derivatives thereof. 2.The process according to claim 1 wherein the host microorganismcomprises E. coli.
 3. The process according to claim 1 wherein thebacterial mTS comprises a Streptomyces mTS.
 4. The process according toclaim 1 wherein the bacterial mTS comprises a Streptomyuces clavuligerusmTS.
 5. The process according to claim 1 wherein the bacterial mTScomprises cineole-1,8 synthase or linalool synthase.
 6. The processaccording to claim 1 wherein the process results in a monoterpene yieldof at least 100 mg/L_(org) ⁻¹.
 7. The process according to claim 1wherein the process results in a monoterpene yield of at least 300mg/L_(org) ⁻¹.
 8. A microorganism for use in producing a monoterpeneand/or derivatives thereof according to the process of claim
 1. 9. Arecombinant microorganism adapted to conduct the following step:converting geranyl pyrophosphate (GPP) into a monoterpene and/orderivatives thereof by expression of a bacterial monoterpene synthase(mTS).
 10. The use of a bacterial mTS to improve the yield and/or purityof a monoterpene and/or derivatives thereof obtained from GPP.